The DsRed2-ER (#632409) plasmid was purchased from Clontech Laboratories, Inc. (Mountain View, CA). GFP-Fundc1Δ7-48 mutation plasmid was generated using Quikchange ӀӀ Site-directed Mutagenesis Kit (Agilent Cat: 200523). The procedure was followed with the company protocol. The GFP-Fundc1 plasmid (Origene Cat: RG208211) was used to delete region 7-48. The resulting plasmid was sequenced and confirmed with T7 primer. Cardiomyocytes were transfected with plasmids using Lipofectamine 2000 (#11668-019, Life Technologies, Carlsbad, CA), according to the manufacturer’s protocol.
Fundc1 siRNA (#sc-145273) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas). Itpr2 siRNA (#n435663) was purchased from Life Technologies. For gene silencing of Fundc1 or Itpr2, cardiomyocytes were transfected with 10 μmol/L siRNA using Lipofectamine®RNAiMAX (#13778150, Life Technologies, Carlsbad, CA) according to the instructions provided by the supplier.
The mouse neonatal cardiomyocytes or H9c2 myoblasts were infected with adenovirus encoding Fundc1 (#197503A and #281786A, Applied Biological Materials Inc., Canada) in normal culture medium for 48 h. An adenoviral vector encoding beta-galactosidase (β-Gal) (#000197A, Applied Biological Materials Inc., Canada) was used as a control. Adenovirus encoding constitutively active Ampk mutants (ad-Ampk-CA) was generated as described earlier30 (link).